Protein C and protein S deficiencies contribute to morbidity and mortality in men and mice. Recombinant acfivated protein C (APC) therapy reduces mortality in adult severe sepsis patients. There is a major need for new insights into the physiologic and pharmacologic mechanisms of action of APC and protein S. To establish in vivo proof of principle for such mechanisms. Project 1 uses genetically modified mice, murine injury models, and novel recombinant murine proteins and involves extensive collaborafions with Drs. Ruggeri and Ruf APC exerts two distinct activities: (1) anticoagulant activity and (2) direct beneficial effects on cells comprising a variety of cytoprotective actions. This latter activity is critical for mortality reduction by APC in murine sepsis models. The current paradigm for APCs cell signaling involves binding of APC by endothelial protein C receptor (EPCR) combined with protease activated receptor-1 (PARI) proteolytic activation. We found that there is another signaling pathway initiated by APC that involves ligation of apolipoprotein E Receptor 2 (apoER2), signaling via the adaptor protein. Dabi, and Src-family kinases with downstream acfivation of the PI3K-Akt survival pathway. Engineering of murine APC and apoER2 mutants will allow interrogation ofthe protein surfaces that mediate binding and signal inifiation by APC:apoER2 interactions and will provide reagents for in vivo proof of principle studies for mechanisms of APCs acfion in murine sepsis. Studies of mice genefically modified in apoER2 and Dabi will establish whether apoER2 and Dabi mediate APCs mortality reducfion acfivities in sepsis. Protein S deficient mice will be subjected to thrombofic provocafion and treated with combinations of recombinant wild type and mutant murine protein S and APC or other agents to define the relafive efficacies for protein S antithrombotic activity that is either dependent on APC or independent of APC. Novel Principles that are established by these preclinical animal model studies may ultimately be translated into diagnosfic or therapeufic advances involving the protein C and protein S systems. RELEVANCE (See instmctions): Protein C or protein S deficiency contributes to excessive blood clotting. Recombinant acfivated protein C (APC) is FDA-approved for reducing death in adult severe sepsis, but we don't understand how this drug works. To gain new insights, we will use engineered APC and protein S plus mice that are genefically modified. Novel principles that are established by these preclinical animal model studies may ulfimately be translated into diagnosfic or therapeutic advances involving the protein C and protein S systems.